Inhibitors of HIV reverse transcriptase (RT) polymerase activity are routinely used for therapeutic purposes, yet no inhibitor of the protein's ribonuclease H (RNH) activity is available for clinical application. Information on how RNH inhibitors (RNHI) interact with RT, at an atomic level, is scarce, and this may contribute to the difficulty in targeting the ribonuclease for therapeutic purposes. Only recently were crystal structures of RNH active-site inhibitors in complex with RT or an RNH fragment published, and structural analysis of non active- site RNHI has not been carried out. Part of the difficulty in understanding the atomic mechanisms of RNH inhibition relates to the conformational flexibility of RNH: the RNH domain is known to have relatively large flexible regions that are important for substrate recognition and the enzymatic reaction. Yet, how this flexibility influences inhibitor binding, and vice versa, has not been studied in a systematic manner. The goal of the proposed research is to investigate RNHI interactions with an RNH fragment and with full-length RT, at an atomic level, in solution. For this purpose, solution NMR spectroscopy, in concert with biochemical and virological studies, will be carried out on RNH, using RNHIs as probes to detect conformational changes. The proposed research will provide insight into RNH domain conformational changes upon RNHI interaction, knowledge of where RNHIs interact with RNH in solution, and an understanding of how RNHI binding pockets become conformationally stable. Such information will be useful for determining whether RNH is a feasible target for drug development. We will complete the following specific aims: (1) Characterize RNH conformational changes upon interaction with active-site RNHIs in solution, (2) Identify the structural mechanism of allosteric inhibition and the allosteric RNHI interaction site on RNH, (3) Examine the interactions of RNHIs with full-length RT in solution, and (4) Validate the structural findings using site-specific mutagenesis coupled with in vitro and in vivo assays.